Stable vitamin b12-containing solution



United States PatentOfi 2,8233%? Patented Feb. 11, 12958 2,823,167STABLE VITAMIN Il -CONTAINING SOLUTION Harold L. Newmark, Lynhrook, N.Y., assignor to The This invention relates to'a new and improved vitaminB composition and a new-and. improved method of stabilizing vitamin BVitamin B is described-in'tlre United "States Pharmacopeia, XIV.Edition; page 660 and on'pages 1002-1003 of the sixth edition"(published-irr'1952)" of" The Merck Index, publishedby M-erck*&'Co'.-,Inc:

It is designated by; numerous other'names,;which*'are mentioned in saidpage 1002:

In solid condition, cyanocobalamin-is'irr the formof hygroscopic;dark-red crystals which are stable in solid form. One gram thereofdissolves in about 80 ccof water at 25 C.

Said vitamin B is substantially stable in solid form and in aqueoussolution if; dissolved without other vitamins, with maximum stability ina pH- range of4.5- to: 5.

When: theaqueous. solution, in addition: to said vitamin B contains.other dissolved vitamins; particularly dissolved vitamin C (ascorbicacid), B; and niacinamide, the dissolved vitamin B is unstable.

In making an aqueous'solution. of vitamin B with other vitamins, it is;known to add gvarious additional ingredients, such as buffers,preservatives, flavoringagents, etc. These additional ingredients mayinclude liver extract which may contain iron in combined form, ormineral salts whichmay contain iron in combined form. The liver extractor said mineral. salts are added for therapeutic purposes.

The technical literature states that solid or undissolved crystallinevitamin B is stable in thepresence of ferricv ammonium citrate andferric'glycerophosphate, but that vitamin B is incompatible withferroussulfateand is incompatible with vitamin C, thiamin plusniacinamide and anypotent reducingsubstance.

According tothis invention, it has been discovered that.

a mixed aqueous solution of vitamin B and another vitamin Or othervitamins, particularly amixedaqueous solution containingvitamin Band.vitamin C,.can be stabilized by all iron compounds orsalts.Thezdegreeof stabilization of vitamin B achieved. depends. ontheparticular iron. compound or salt used, the concentration of-ironcompound or salt:used, especially in comparison with the concentration.of. vitamin. B which must be stabilized; and thenature. andconcentrations of the other substances present in the solution- Thesestabilizing' iron compounds. or. salts-are used inthis invention. asstabilizing agents, and not as therapeutic agents, and, accordinglytheir concentration is below the therapeutic level. This is importantand. representsthe essence of this invention inview of the following.Ferrous .compoundssin solutionwithvitamin B cause. rapid destruction. of"vitaminB when present in therapeutic concentrations. This hasbeenverified by several investigators... Ferrouscompounds in .therapeuticamountsare cQntra-indicatedfor inclusion evenin solid formulations,

incombination with vitamin B3 ,due to the deleterious effect on vitaminBi stability. Yet, in very low concentrations thesesarne ferrouscompounds will stabilize vitamin Bi; imthe presenceof other substancesnormally considered'fleleterious tovit'amin 'B stability (if e.vitam'ir'rC",thiamin andniacinamidtfii These-concentrations are farbelow the amounts of iron commonly used in preparations to. supply theminimum daily therapeutic dose of iron (i. e., for hypochromic anemias,etc.). My invention is based'on the discovery of'this unexpected" andsurprising phenomenon.-

7 These stabilizing iron compounds. or salts of. the invention areexemplified, without'limitation thereto; by the following: ironpeptonate,.ferric: ammonium citrate, ferric chloride; ferrousgluconate;ferric glycero phosphate, ferric sulfate,.ferrous sulfate,ferric oxide, ferrousoxide, ferric or'ferrous complexes ofsuch'substances as" ethylene diamine tetraacetic acid' and its salts.

It-has-also' be'err discovered thatthe stabilizing effect begins at acertain minimumconcentration of'iron' ('calculated as atomic iron) inthe solution; that said stabiliz ingrefict lincreases up. to. a. certainmaximum concentrati'on; of -iron,:calt:ulated as. atomiciron, and. thatthestabilizing efiect; decreases. whensaid maximum concentration isexceededgqalsot calculating the. iron. as.v atomic iron. Also, thesta'bilizihgeffect. depends upon. the respective ironcomponndQ-fiinaddition. to. its concentration. The stabilizingreffot.also..dep.ends. upon the. dissolved ingredients other thanzdissolvcdvitamin. B which are. dissolved in thersolutiom- The iron saltsoncompounds. may. have. a respective therapeuticzefict,lbut.theiiconcentratiorr thereof, as above noted, is below theconcentration for the respective therapeutic eifecu. I

Thesvita-min B maybe admixed with the stabilzing agent in solid form,and themixture may be: dissolved in water. for use.

Hence the invention. includes undissolved mixtures,- as well assolutions of said mixtures.

. All .pH..measurementsmentioned herein are made at 20. C..'30: C.,.,,bymeansof the glass electrode.

Withoutlimitation thereto, the invention is. illustrated by thefollowing} examples which show' some; embodiments of; .and some bestwaysfor carrying out the inventlOI't'.

In' the rfollowingqExamples' l-9 twoaqueous solutions of. thefollowing-composition were. used:

SOEUTIONI" Each 5 cc. of this solution contained: Vitamin A 3000 U. S.P. units. Vitamin D 1000 U. S. P. units.

(cyanocobalamin)* 61' mcg. VitaminiC (ascorbic acid) mg. Niacina-mide10mg.

With the exception of the vitamin. B concentration,

this=solutionhad the same composition as the above described Solution I.Each 5 cc. of Solution II contained 3--mcg; of vitamin B,(.cyanocobala1nin:) ,,i. e. half. of the :amount, present in SolutionI.

These solutions were subdivided andiron peptonate or ferric. ammoniumcitrate were added in the amounts stated hereinafter to separateportions, which were then stored at 37 C."Ifra' p'eriodof 'three Weeks.After this period the vitamiri Bm concentration of thesol'uti'ons Wasassayed-bythe?microbiologicalmethoddescribed in U; S; P. XIV3d"Supple'm'ent'.' This method, as normally used, is accurate withane'rrorz'ofi Iii-20%; which is'a =reasbnable error for amicrobiological assay of this type.

Table 1 Table 3 B1: assay Bu assay No. of Solution Amount of after No.of Solution Amount of after Example used Stabilizer used Stabilizer,storage Example used Stabilizer used Stabilizer, storage mgJcc. at 37 C5 mgJcc. at 37 0.,

meg/5 cc meg/cc.

I iron peptonate 0.1 7. 05 IV iron peptonate 0.1 25. 2 I d 1.0 5. 46 IV-do 1.0 19.1 I do 10. 0 6. 50 IV 10. 0 18.1 I ferric ammonium elt- 0. 14. 35 10 IV iron ammonium 0112- 0.1 19. 9

rate. I d0 1.0 2.58 IV 1.0 16.5 I do 10. 0 2. 04 IV 10.0 14. 8 II ironpeptonate- 10. 0 3.19 IV 0 1 none 1. 78 II de 0 In Examples 20-22, asolution denoted hereinafter In Examples 10-12, a solution containing ineach co. the following vitamins were used:

Vitamin E (mixed tocopherols) 1.67 mg.

This solution also contained suitable flavoring agents such asglycerine, propylene glycol, saccharin, raspberry flavor; assolubilizerfor vitamin A, D and E: polyoxy ethylene sorbitan monolaurate, orpolysorbate 80, and, as preservative: methyl parahydroxy benzoic acidand/or sodium benzoate.

The solvent used was a mixture of glycerol, propylene glycol anddistilled water. To the solution thus prepared vitamin B(cyanocobalamin) was added to produce a concentration of approximately14 mcg. per cc. The resulting liquid, which is denoted hereinafterSolution III was subdivided into several portions, to which thestabilizers listed in Table 2 were added and which were then stored at37 C. for three weeks. After storage the vitamin B content was assayedwith the following results:

In Examples 13-19 an aqueous solution denoted hereinafter Solution IVwas used, 1 cc. of which contained the following vitamins:

B 100 mg. B 1 mg. B 2 mg. B (cyanocobalamin) 23 mcg. Niacinamide 100 mg.Pantothenic acid (panthenol) 10 mg. Benzyl alcohol 1.5% (based onweight/volume).

This solution was subdivided into several portions, to

.which the stabilizers listed in Table 3 were added. The solutions werestored at 37 C. for three weeks and after 37 C. for three weeks.

Solution V was used, which contained the following ingredients in eachone cc.:

Vitamin B 100mg.

Vitamin B 1mg.

Vitamin B 2mg.

Niacinamide 50 mg.

Panthenol 10 mg.

Vitamin B (cyanocobalamin) 15 mcg.

Vitamin C 100mg.

Buffers, preservatives, etc 6% (based on weight/ volume).

Distilled water q. s. 7

As bufiers and preservatives, sodium citrate (2% w./v.), benzyl alcohol(1% w./v.) and gentisic acid ethanolamide (3% wQ/v.) were present in thesolution. The

initial assay ofthe solution was 16.1 mcgm. of vitamin B per cc.

EXAMPLE 20 To a portion of Solution V, 0.5 mg./cc. of iron peptonate wasadded and resulting solution was stored at 37 C. for three weeks. Aftersuch storage, the solution showed a vitamin B 5 assay of 15 mcg./cc.Since this solution originally had 15 micrograms of cynocobalarnin percc., the microbiological assay showed a concentration of 100% ofpotency.

EXAMPLE 21 To a portion of Solution V, '12 mg./cc. of iron peptonatewere added and the solution was then stored at After storage, a vitaminB assay of 7 mg./ cc. wasfound. Hence in this case, the value of thecyanocobalamin found in the microbiological assay was substantially 50%of the original concentration.

EXAMPLE 22 After storage at 37 C. for three weeks without any addition,a portionof Solution V showed a vitamin B assay of less than 1 meg/cc.

In the preceding Examples 20-22 there is a demonstration of the decreasein stabilization of vitamin B achieved when the concentration of ironcompound morenearly approaches therapeutic levels. Solution V, whichcontains ascorbic acid, thiamin and niacinamide, etc., is normallyconsidered a poor medium for vitamin B stability, a view verified byExamples 22. Addition of a small amount of iron compound (0.5 mg./cc. ofiron peptonate) as in Example 20, produced a solution of good vitamin,B1 stability. A high concentration of the same iron compound, as used inExample 21, showed a sharply decreased vitamin B stability in comparisonwith Example 20. It is to be noted that the atomic iron concentration ofExample 21 was 2 mg./cc. The normal human dose of this preparation is 1cc. Therefore, this preparation supplies one-fifth the minimum dailyadult iron requirement per dose. Yet, this concentration of iron, inrelation to the other ingredients, produced a less that time theirvitamin B content was assayed. Th

. results are shown in the following table:

Vitamin A. (.palmitate.) 833.0.U. S. P.1UnitS. VitaminD 2.00.011. S.-P."Vitamin B. 3.33 mg.

Vitamin. B 0.83. mg.

Vitamin B 1.67 mg.

Vitamin C 100mg. Niacinamide 16.67 mg. Pantothenic acid (as panthenol)mg.

Choline chloride 12 mg.

Inositol Tmg.

Vitamin E (mixed,tocophero1s). 1.67 mg.

The. solvent was a mixture of glycerol, propylene glycol and distilledwater;

The resulting solution was divided into 3 porti'ons-, and to theseportions vitamin B was added as followsr (a) To the first portion, 10mcg: per cc. of vitamin B activity, type S (Merck) was added, i. e.vitamin B consisting of a semi-refined. B fermentation product, whoseactivity is due entirely to cyanocobalaminand which is blended withinert ingredients suitable for bulk handling to a concentration of 1 mg.vitamin B activity per gram ofgross weight.

(b) To the. second portion, 10 .mcg. per cc. of vitamin B activity wasadded, in the form of oral grade solids (.Calco), consisting of aconcentrate in semi-purified state of B activity components obtainedfrom microbiological' fermentation, suitable for oral use and blendedwith inert ingredients to an activity of 1 mg. of B per gram of grossweight. This vitamin B activity is composed of approximately 75-80% ofhydroxy cobalamin, the balance being predominantly cyanocobalamin.

(c) To the third portion, 10 mcg. per cc. of vitamin.

B activity was added inthe form of U. S. P. crystalline cyanocobalamin.

Each of these portions (a), (b') and (c), was again subdivided into 3parts and iron peptonate was added to some, in the amounts listed below;Assayswere taken at the time of preparation and after'storage for 3Weeks at 37 C. The results were as follows:

The above data showthat. 1" mg. per cc. of iron peptonate. stabilizeseach of the threetypes -of-vitamin B equally well in the preparationherein described.

A multivitamin solution containing in each cc. the followingingredients, was prepared:

Vitamin A, (-palmitate) 830.0'U.'..S.1P'. units. Vitamin D; 2000 U. S.P. units. Vitamin B 3.33 mg.

Vitamin B 0.83 mg.

Vitamin B 1.67 mg.

Vitamin C 100 mg. Niacinamide 16.67 mg. Pantothenic acid (as panthenol)5 mg.

Vitamin E (mixed tocopherols) 1.67 mg.

In this preparation, which is similar to that, used in the aboveExamples 10-12, with the omission of choline chloride and inositol, thesameflavoring and solvent as incExamples 1-0i-12: were used:

To thisipreparation vitamin B ('cyarrocolialamin): was added. to producea concentration of" about 4' meg/cc. This solutiondenoted;hereinafterSolution V, was divided into 13:; portions; and tothese portions iron compounds were added-,fas described below. Afterstorage for 3 weeks at 37 C. the. vitamin B was assayed, and thefollowing results were obtained:

Table 5 Bu assay Amount of 'Amount'of after Stabflizer'used (if any)"Stabilizer, Fe per cc., 3 weeks mg./cc. meg/cc. storage at 37 0.,meg-.lcc;

(a) none 0 0 Eb; gerrle clglorlfiethb f 1 200 2:24

c erriec an owl ee pep- ..tone

2 200 (ti) ferrous gluconate 1 116 1:89 (a) Ferrous gluconate with beefpeptone 1 116 1:80 (1') ferric glycero phosphate. 1 180 2. 84 (g ferricammonium citrate. 1 2: 24 (h). ferric sulfate 1 280 3. 35 (ig ferroussulfate" 1. 200 2. 76 (j ironpeptonate. 0.01 156 0 (k) iron'peptonate0.1 16 0 (1) iron peptonate 1. 0. 160 1.38 (m) ironrpeptonate 10.0 1,600 4.18

The abovev results demonstrate that all of the iron compounds used havea stabilization effect on vitamin B in this preparation, whichcontainsascorbic acid as well asthiamin, nia-cinamide, and other vitamins. Wherenostablizer is used the vitaminiB decomposes rapidly. The, results. withiron peptonate (j through m) indicate that a.minimum amount of iron isrequired to achieve vitamin B stabilization in this preparation, andthat this stabilization effect increases. as the amount of ironincreases wthin the range of concentrations tested in this experiment-Generally, with only a few exceptions,. the above data indicate anincreasing stabilization of vitamin B in this preparation as the ironconcentration increases within the limited range of concentrationstested. This is true regardless of the type of iron compound used.

In certain types of multivitamin preparationsit may be deemedinadvisable to add appreciable amounts of iron compounds which yieldappreciable amounts of ferricor ferrous ionsin solution (e. g., ferricchloride). This is due to the known adverse eifect of ferric ions. onthe stability of certain vitamins (e. g., vitamin A, pyridoxinehydrochloride, etc.). Howevenit is also well knownthat iron. may stillbe present in such solutions. ifpresent in the form of complexes orchelates. These'bindferric and/or ferrous ions into soluble forms whichare only very slightly dissociated into the ionic forms of, iron.Several of the stabilizers used in the above: examples are illustrationsof. such complexes (e. .g., ferric. ammonium citrate, iron peptonate,etc.). One of the. most efiective of such chelating agents is-ethylenediamine. tetra-acetic acid and its salts (called EDTA. or versene(R)).-.So effective, is this agent that it hasbeen recommendedfor use asastabilizer for B complex,.ascorbic.acid, andother vitamin, preparationsto prevent the. breakdown of these vitamins in the presence of ioniciron. Itis often .used. as a stabilizer in such preparatons because ofths.proper.ty. However, even in the presence of EDTA, iron compoundswill still stabilize vitamin B in multivitamin preparations. Thisunexpected and surprising effect is of great practical importance, sinceit enables the use of iron compounds in multivitamin preparations tostabilize vitamin B and, yet, the iron is so efiectively combined in thechelate form that there is excellent stability of the other vitamins inthe preparation. This is illustrated in the following Example 25.

I EXAMPLE 25 A solution identical withthe composition used in Solution Iand II was prepared, except that the vitamin B '3 weeks at 37 C. thevitamin B was assayed and the following results were obtained:

Table 6 B19 assay Amt. of Amt. of Amt. of after Portion Stabilizer usedStabilizer, Fe per 00., EDTA, 3 Weeks (it any) mg./ec. mg./cc. mg./cc.at 87 0.,

' mcgmJ None 0 0 1.1 do 0 0 1 0.8 Ferric Chloride 0. 5 0. 1 1 3. 6

Hexahydrate. do 0.5 0.1 0 3.7 Iron Peptonate 0. 6 0.1 1 3. 7 do 0.6 0.10 4.3 Ferric Ammo- 0. 7 0. 1 1 4. 1

mum Citrate. do 0.7 0.1 0 4.0

From the above data in Table 6jis' canbe seen that the vitamin B in thispreparation (which contains vitamins A, C, D, thiamin, niacinamide,andIother vitamins) is unstable without the addition of any ironcompound. Addition of any of the three types of iron compound used(completely ionized as ferric chloride, partly ionized as ferricammonium citrate, or non-ionic as iron peptonate) will materially aidthe stability of vitamin B Addition of EDTA without iron (portion b)does not stabilize the preparation. The simultaneous addition of an ironcompound with EDTA does not appreciably reduce the vitamin B stabilizingeffect of the iron compounds. It is to be noted that the amounts of EDTAadded in each case are in excess of that required to chelate the entireamount of iron added. In portions (c), (e), and (g) the EDTA added wassufiicient to protect the other vitamins from iron-produced breakdown,so that no appreciable decrease of potencies of vitamin A, thiamin,pyridoxin, ascorbic acid, riboflavin, and other vitamins took place. Thetests previously stated herein apply to solutions whose solvent includeswater, and in which the original concentration of the dissolved ascorbicacid is greater than the original concentration of the dissolvedcyanocobalamin. In the tested solutions, the dissolved ascorbic acid wasin sufficient concentration to produce decomposition of the dissolvedcyanocobalamin, thus resulting in substantial or complete destruction ofthe cyanocobalamin, when determined by microbiological assay at the endof a storage period of three weeks at 37 C., in the absence of asuitable proportion of added stabilizing agent. When the solution had astabilizing iron compound in suitable stabilizing concentration and thesolution was tested by said microbiological assay at the end of saidstorage period of three weeks at 37 C., the value of the cyanocobalaminwhich was determined by said microbiological assay, was at leastone-half of the original concentration. Alternatively stated, the valueof the cyanocobalamin which was determined by said microbiologicalassay, in the presence of a suitable concentration of the stabilizingiron compound, was at least twice the lower value in the absence of suchsuitable concentration.

, The solutions to which a stabilizer according to the present inventionis added have a pH in the range of 3 to 7. For example, the pH of theabove described solutions is as follows:

Solution I 5.0 Solution II 5.2 Solution III 5.1 Solution IV 4.3 SolutionV 4.5 Solution VI 5.0 Solution V" 5.0

' definition in Federal Register of November 22, 1941, title 21, Foodand Drugs, chapter I, part 125, page 5923, paragraph 52.

What is claimed is:

1. An aqueous solution which includes dissolved cyanocobalamin anddissolved ascorbic acid, the original concentration of said ascorbicacid being greater than the original concentration of saidcyanocobalamin, said dissolved ascorbic acid being in sufficientoriginal concentration to induce decomposition of said dissolvedcyanocobalamin in a cyanocobalamin-destroying reaction, in the absenceof a stabilizing agent; said solution having an added stabilizing agentdissolved therein, said stabilizing agent being an iron compound, saidiron compound being soluble and substantially non-toxic in theconcentration present in said solution, the elemental iron content insaid stabilizing iron compound being in the range of substantiallyseventeen micrograms to seventeen-hundred micrograms per cubiccentimeter, said selected concentration of said iron compound beingbelow a daily dosage of ten milligrams of elemental iron.

2. A solution according to claim 1, said solution having an originalconcentration of dissolved cyanocobalamin of at least six-tenths of amicrogram per cubic centimeter.

3. A solution according to claim 2, the original concentration of thedissolved ascorbic acid being in excess of one thousand times theoriginal concentration of the cyanocobalamin.

References Cited in the file of this patent UNITED STATES PATENTS SkeggsFeb. 5, 1952 Weidenheimer Nov. 30, 1954 OTHER REFERENCES UNITED STATESPATENT OFFICE CERTIFICATE OF CORRECTION Patent No, 2,823,167 February11, 1958 Harold L. Newmark It is hereby certified that error appears inthe printed specification of the above numbered patent requiringcorrection and that the said Letters Patent should read as correctedbelow.

Column 2, line 53, 1 after "lemon" strike out the comma and insertinstead a semi-colon; line 54, after "80" strike out the comma andinsert instead a semi-colon; line 56, after "benzoate" insert asemi-colon; line '72, after "type." insert The results are indicated inTable I. column 3, lines 16 and 1'7, for "containing in each cc. thefollowing vitamins were used:" read Was used, With the followingcomposition in each one cc. I column 4, line 47, for "'7 mg./cc." read'7 mcg/cc. line 61, for "Examples" read Example column 6, line '7, for"Solution V" read Solution VI Signed and sealed this 27th day of May1958.

(SEAL) Attest:

KARL AXLINE ROBERT c. WATSON Attestlng Officer 7 Comnissioner of Patents

1. AN AQUEOUS SOLUTION WHICH INCLUDES DISSOLVED CYANOCOBALAMIN ANDDISSOLVED ASCORBIC ACID, THE ORIGINAL CONCENTRATION OF SAID ASCORBICACID BEING GREATER THAN THE ORIGINAL CONCENTRATION OF SAIDCYANCOBALAMIN, SAID DISSOLVED ASCORBIC ACID BEING IN SUFFICIENT ORIGINALCONCENTRATION TO INDUCE DECOMPOSITION OF SAID DISSOLVED CYANOCOBALAMININ A CYANOCOBALAMIN-DESTROYING REACTION, IN THE ABSENCE OF A STABILIZINGAGENT, SAID SOLUTION HAVING AN ADDED STABILIZING AGENT DISSOLVEDTHEREIN, SAID STABLIZ-ZING AGENT BEING AN IRON COMPOUND, SAID IRONCOMPOUND BEING SOLUBLE AND SUBSTANTIALLY NON-TOXIC IN THE CONCENTRATIONPRESENT IN SAID SOLUTION, THE ELEMENTAL IRON CONTENT IN SAID STABILIZINGIRON COMPOUND BEING IN THE RANGE OF SUBSTANTIALLY SEVENTEEN MICROGRAMSTO SEVENTEEN-HUNDRED MICROGRAMS PER CUBIC CENTIMETER, SAID SELECTEDCONCENTRATION OF SAID IRON COMPOUND BEING BELOW A DIALY DOSAGE OF TENMILLIGRAMS OF ELEMENTAL IRON.